Shigella species are the major pathogenic bacteria causing bacterial diarrhea in developing countries, which infect 164.7 million people and lead to 0.11 million deaths per year, most of which are children under 5 years old (Kotloff, K. L., J. P. Winickoff, B. Ivanoff, J. D. Clemens, D. L. Swerdlow, P. J. Sansonetti, G. K. Adak, and M. M. Levine. 1999. Global burden of Shigella infections: implications for vaccine development and implementation of control strategies. Bull World Health Organ 77:651-66). Among the four serogroups of Shigella, Shigella flexneri is the predominant serogroup that affects the low-income population.
Shigella flexneri is further classified into various serotypes according to the different 0-antigen structures. To date, at least 15 serotypes have been reported, i.e. 1a, 1b, 1c, 2a, 2b, 3a, 3b, 4a, 4b, 5a, 5b, X, Xv, Y, and F6 (Simmons, D. A., and E. Romanowska. 1987. Structure and biology of Shigella flexneri O antigens. J. Med. Microbiol. 23:289-302; Stagg, R. M., S. S. Tang, N. I. Carlin, K. A. Talukder, P. D. Cam, and N. K. Verma. 2009. A novel glucosyltransferase involved in O-antigen modification of Shigella flexneri serotype 1c. J. Bacteriol. 191:6612-7; Ye, C., R. Lan, S. Xia, J. Zhang, Q. Sun, S. Zhang, H. Jing, L. Wang, Z. Li, Z. Zhou, A. Zhao, Z. Cui, J. Cao, D. Jin, L. Huang, Y. Wang, X. Luo, X. Bai, P. Wang, Q. Xu, and J. Xu. 2010. Emergence of a new multidrug-resistant serotype X variant in an epidemic clone of Shigella flexneri. J. Clin. Microbiol. 48:419-26). Serotyping has long been use to characterize isolates for epidemiological purposes
The LPSs of all Shigella flexneri serotypes except F6 have the same polysaccharide backbone consisting of repeating tetrasaccharide units, and serotype Y has the basic tetrasaccharide backbone (Simmons, D. A., and E. Romanowska. 1987. Structure and biology of Shigella flexneri O antigens. J. Med. Microbiol. 23:289-302). Modification by glycosylation and/or acetylation of different sugars on the backbone gives rise to various type-specific antigenic determinants (e.g., I, II, III, IV, V, and VI), group-specific antigenic determinants (e.g., 3,4; 6; and 7,8), and antigenic determinant 1c (Stagg, R. M., S. S. Tang, N. I. Carlin, K. A. Talukder, P. D. Cam, and N. K. Verma. 2009. A novel glucosyltransferase involved in O-antigen modification of Shigella flexneri serotype 1c. J. Bacteriol. 191:6612-7).
Three genes (gtrA, gtrB, and gtr[type]) are responsible for glycosylation modifications on Shigella flexneri. The first two genes are highly homologous and interchangeable, whereas the third gene gtr[type] is unique, and encodes serotype-specific glycosyltransferases (Allison, G. E., and N. K. Verma. 2000. Serotype-converting bacteriophages and O-antigen modification in Shigella flexneri. Trends. Microbiol. 8:17-23; Stagg, R. M., S. S. Tang, N. I. Carlin, K. A. Talukder, P. D. Cam, and N. K. Verma. 2009. A novel glucosyltransferase involved in O-antigen modification of Shigella flexneri serotype 1c. J. Bacteriol. 191:6612-7). Gtr genes specific to type-antigens I, II, IV and V, group antigen 7,8 and antigen 1c are gtrI, gtrII, gtrIV, gtrV, gtrX and gtrIC, respectively (Adams, M. M., G. E. Allison, and N. K. Verma. 2001. Type IV O antigen modification genes in the genome of Shigella flexneri NCTC 8296. Microbiology 147:851-60; Adhikari, P., G. Allison, B. Whittle, and N. K. Verma. 1999. Serotype 1a O-antigen modification: molecular characterization of the genes involved and their novel organization in the Shigella flexneri chromosome. J Bacteriol 181:4711-8; Guan, S., D. A. Bastin, and N. K. Verma. 1999. Functional analysis of the O antigen glucosylation gene cluster of Shigella flexneri bacteriophage SfX. Microbiology 145: 1263-73; Huan, P. T., D. A. Bastin, B. L. Whittle, A. A. Lindberg, and N. K. Verma. 1997. Molecular characterization of the genes involved in O-antigen modification, attachment, integration and excision in Shigella flexneri bacteriophage SfV. Gene 195:217-27; Mavris, M., P. A. Manning, and R. Morona. 1997. Mechanism of bacteriophage SfII-mediated serotype conversion in Shigella flexneri. Mol Microbiol 26:939-50; Stagg, R. M., S. S. Tang, N. I. Carlin, K. A. Talukder, P. D. Cam, and N. K. Verma. 2009. A novel glucosyltransferase involved in O-antigen modification of Shigella flexneri serotype 1c. J Bacteriol 191:6612-7). The gtr genes are carried by prophages integrated in the genome of host bacteria. O-acetylation, which confers group-antigen 6 and/or type-antigen III on strains of serotypes 1b, 3a, 3b and 4b, is mediated by the oac gene carried in bacteriophage Sf6 (Clark, C. A., J. Beltrame, and P. A. Manning. 1991. The oac gene encoding a lipopolysaccharide O-antigen acetylase maps adjacent to the integrase-encoding gene on the genome of Shigella flexneri bacteriophage Sf6. Gene 107:43-52; Verma, N. K., J. M. Brandt, D. J. Verma, and A. A. Lindberg. 1991. Molecular characterization of the O-acetyl transferase gene of converting bacteriophage Sf6 that adds group antigen 6 to Shigella flexneri. Mol. Microbiol. 5:71-5). Strains of different serotypes carry one or more serotype-specific prophages that encode different specific 0-antigen modifications genes (see FIG. 1).
Currently, the method for routinely identifying Shigella flexneri serotypes is the slide agglutination method using rabbit-originated antiserum raised against Shigella flexneri-specific type- and group-factors. Commercially available diagnostic sera have been widely used in microbiology laboratories. However, such a method has some disadvantages. Firstly, to identify the serotype of one Shigella strain, the slide agglutination method requires as many as 10 reaction tests, using antibodies against type-antigens I, II, III, IV, V and VI, antibodies against group-antigens 3,4 and 7,8, and the monoclonal antibody against serotype 1c (MASF1c), respectively (Stagg, R. M., P. D. Cam, and N. K. Verma. 2008. Identification of newly recognized serotype 1c as the most prevalent Shigella flexneri serotype in northern rural Vietnam. Epidemiol Infect 136:1134-40; Talukder, K. A., Z. Islam, M. A. Islam, D. K. Dutta, A. Safa, M. Ansaruzzaman, A. S. Faruque, S. N. Shahed, G. B. Nair, and D. A. Sack. 2003. Phenotypic and genotypic characterization of provisional serotype Shigella flexneri 1c and clonal relationships with 1a and 1b strains isolated in Bangladesh. J Clin Microbiol 41:110-7; Ye, C., R. Lan, S. Xia, J. Zhang, Q. Sun, S. Zhang, H. Jing, L. Wang, Z. Li, Z. Zhou, A. Zhao, Z. Cui, J. Cao, D. Jin, L. Huang, Y. Wang, X. Luo, X. Bai, P. Wang, Q. Xu, and J. Xu. 2010. Emergence of a new multidrug-resistant serotype X variant in an epidemic clone of Shigella flexneri. J Clin Microbiol 48:419-26). Secondly, visual assessment of the slide agglutination reactions may deliver incorrect readings. Thirdly, expensive antiserum kits limit the application of such a method in laboratories in developing countries.
Therefore, development of a rapid, specific identification method based on biomolecular technologies such as PCR will be significant for timely and accurate identification of Shigella flexneri serotypes.